PROJECT SUMMARY Langerhans cells (LCs) are skin-resident dendritic cells (DCs) expressing the C-type lectin Langerin (CD207) that mediate both adaptive immunity and immune tolerance in skin and are involved in various types of skin diseases. Adult LCs are originated from embryonic yolk-sac-derived macrophages and fetal liver monocytes in the steady state. Interestingly, LCs could also be derived from the bone marrow or peripheral monocytes and repopulate the skin under inflammatory conditions. However, due to the lack of molecular profiles at individual LC level, a significant gap remains in our understanding on how a single CD207+ epidermal LC population can induce both immunity and tolerance. Fortunately, new technologies such as the single-cell RNA-sequencing (scRNA-seq) can evaluate cell-to-cell transcriptomic variation, while the single-cell assay for transposase- accessible chromatin using sequencing (scATAC-seq) can assess the epigenomic heterogeneity at single-cell resolution in an unbiased manner. Recently, we identified two major LC subsets in mice, ATF3+Bal2a1b- (mLC1) and ATF3-Bal2a1b+(mLC2) subsets, and three major LC subsets in human including ATF3+ (hLC1) subset using scRNA-seq. We also found in ATF3 knockout mice that lack of ATF3 enhances LC maturation and promotes LCs-induced Th1 and Th17 cell differentiation suggesting immune suppressive function induced by ATF3+LC1. Hence, these preliminary data support our hypothesis that LCs are heterogeneous consisting of distinct subsets with different immune functions. Our objective is to use single-cell analysis platforms plus the LC fate-mapping and mutation mouse models to further validate this. We will pursue two Specific Aims in the R61 phase: Aim 1) Characterize the gene signatures and regulatory elements of mLC1 and mLC2 by profiling LCs during embryonic, young, and aging development at steady-state and repopulated LCs at inflamed state using scRNA-seq and scATAC-seq; Aim 2) Generate ATF3negEGFP reporter mice to fate-map ATF3+LC1 embryonic development and the dynamic change of ATF3+LC1 and ATF3-LC2 subset at steady state during adult and aging development and at inflammatory state and functionally characterize LC subsets in vitro by sorting ATF3EGFP+ LC1 and ATF3- LC2 cells and rederiving ATF3.loxp mice, which will be crossed with hLangerin-Cre mice to generate LC- specific/time-induced ATF3KO for in vivo functional study. In the R33 phase, we will pursue the following Specific Aim: Aim 3) Functionally characterize ATF3+LC1 subset in vivo using LC-specific ATF3 deletion hLCcre.ATF3KO mice to evaluate the potential immune regulation function of ATF3+LC1 subset in the different disease models, including autoimmune vitiligo, melanoma, and fungi infection models. Our work will uncover the mystery of LC subsets with their specific functions, which will provide new insights into the biology of LCs and lead to the development of LC-based intervention strategies for skin diseases.